Isoelectric Focusing: Theory, Methodology and Applications by Pier Giorgio Righetti (Eds.)

By Pier Giorgio Righetti (Eds.)

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Even with I as low as A/cm2, the field strength in pure water would be 10,000 V/cm, and the Joule heat 1 W/cm3. Any region of pure water will absorb almost all of the available voltage, thereby decreasing the focusing effect and allowing neighboring electrolytes to diffuse with the developing region of pure water. Theoretically, and compatible with the law of pH monotony, a zone of pure water could develop at a neutral pH and, in fact, water is an ampholyte, isoelectric at pH 7. Cationic water (H30+) is repelled from the anode and anionic water (OH-) from the cathode, so that water as a whole tends to concentrate at pH 7.

45). The latter seemed to be an ideal compound for bridging the conductivity gap. 85 to ca. 35, while the ycarboxyl pK will be virtually unaffected or, Ch. 1 pH unit. 5 by as much as 400-500$/0 as compared with conventional carrier ampholytes. 2. , synthesis An interesting variant of the basic synthetic approach of Vesterberg (1969a) has been reported by Charlionet et al. (1979, 1981). It is known that, in classical acrylic acid-type ampholytes, most of the buffering power in the pH range 2-1 1 will have to be found in the different pK values of the amino groups of the polyamino backbone.

The stability of such natural pH gradients is, however, contingent on other factors such as convection and diffusion. The electrical load should not exceed the cooling capacity of the column to prevent convective mixing. Ideally, the conductivity should be uniform throughout the gradient to prevent local overheating in regions containing low levels 16 ISOELECTRIC FOCUSING of ampholyte or ampholytes exhibiting poor conductivity and buffering capacity. Instability of the pH gradient could also arise by anodic oxidation or cathodic reduction of carrier ampholytes.

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